Detection of serum antibody and surface antigen by radial partition immunoassay

ABSTRACT

Disclosed is a method for detection for serum antibody and/or microbial surface antigen by radial partition immunoassay. The method of this invention is applicable to (a) the evaluation of a clinical specimen for identification of a microorganism; (b) antimicrobial sensitivity assays for determination of an efficacious antibiotic for use against a specific microorganism; (c) a semi-quantitative determination of viral surface antigen; and, (d) a quantitative method for the determination of the presence of serum antibodies to microbial antigens. In each of the foregoing applications, the analyte of interest can be immobilized within a porous matrix (solid phase) by simple pipetting of the sample onto the prepared matrix. Appropriate reagents are subsequently applied to the matrix to effect immunochemical interaction of a labeled binding material to the surface antigen (or antibody) of the analyte of interest. The portion of the matrix within which such interaction takes place is termed the &#34;reaction zone&#34;. After a brief incubation period, a suitable wash solution is then applied to the reaction zone of the matrix so as to effect radial partitioning, entirely within the matrix, of bound and unbound labeled binding material. The amount of signal generated by the bound label remaining in the reaction zone is then monitored visually or by an appropriate measuring device.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of copending application Ser.No. 227,664, now U.S. Pat. No. 4,517,288, filed Jan. 23, 1981 in thenames of Joseph L. Giegel and Mary M. Brotherton.

BACKGROUND OF THE INVENTION

1. Field of Invention:

This invention is directed to a method and a diagnostic kit. Morespecifically, the invention described hereinafter relates to a methodfor the detection of serum antibodies to microbial antigens and/or thedirect detection and quantitiation of these microbial antigens by radialpartition immunoassay and a test kit for the performance of such assay.

2. Description of the Prior Art:

(a) A number of the advances in the field of diagnosis of infectiousdisease attributable to microganisms, have been made as a direct resultof automation of the microbial identification process. These advanceshave not only reduced the turn-around time for performance of suchanalysis, but have increased the accuracy of results which can beattained. The major difficulties encountered in the improvement of suchassays is the need to reduce the processing interval for performance ofthe assay. Classically, microbe detection has involved considerabledelay and expense traditionally associated with manual techniques,incubation steps, irreproducibility and unreliability of results. It hasbeen both the hope and objective of the developers of semi- andessentially fully automated systems, that these deficiencies in theclassical methods can be eliminated or minimized through the adaptationof automation to this art.

Semi-automated systems are currently commercially available forperformance of the above type assay. Typically these systems employ somekind of support media (i.e., wells, cups or tubes) into which is placedan aliquot of patient sample. Added to this at different stages aresignal generating reagent(s). After several sets of washings and one ormore periods of incubation, a reaction of this analyte in the samplewith the signal generating reagent(s) occurs. The activity of the signalgenerating reagent(s) is related to the amount of the analyte present inthe sample. The signal measured may be a radiolabeled, a chromogenic ora fluorogenic compound which can be detected by instrumentation; or,observed visually through comparison of color change with a set ofreference colors specific for the given substance being tested.

Since the rapid detection and accurate identification of the microbialspecies is the predominant concern of the clinical microbiolobylaboratory, most of the automated systems which have been introduced aredesigned to reduce the amount of required manipulative steps, and alsoincrease the efficiency of the assay process. Notwithstanding suchadvances, significant limitations in the state of the art still exist.Representation of one such commercially available system is MINITEK®microorganism differentiation system (available from Becton, Dickinsonand Company). This MINITEK system uses a plate containing ten (10)wells, into which are added one or more cartridge-dispensed paper discsimpregnated with a substrate. A microbe colony, isolated from anincubated culture, is also added to the well. The contents of the wellis then covered with a layer of mineral oil to prevent contamination.The contents are thereafter incubated for 18-24 hours. Then, asapplicable, reagents are added and allowed to react with the contents ofthe well; the results of such interaction ultimately being determined bycomparison of color changes in the liquid in the well with theappropriate reference comparator card. One of the more significantlimitations of this MINITEK system as with most others which arecurrently being used, is the delay encountered before results can beobtained. The system requires two extended incubation periods, one togrow the microbe, and a second to allow the sample and substrate toreact. The MINITEK system also is highly labor intensive, requiringseveral manual manipulative steps. Furthermore, the system must be usedwith pure cultures, it being incompatible with specimens taken directlyfrom the patient since the presence of endogenous interactive substancesin the patient sample can interfere with the accuracy of the assay. Theassay results obtained from the tests performed on this system arelimited in precision to a positive/negative determination or to a rangeof color-compared variation.

(b) Antimicrobial Sensitivity Assay techniques have been developed as ameans for rapidly determining the most efficacious drug to be used inthe treatment of a particular infection without the need for obtainingprecise identification of the bacteria causing the infection. Thismethod comtemplates observing which of a set of drugs best inhibits thegrowth in vitro of bacteria contained in a patient sample. the primaryobjective of this screening technique is to provide the physician withan accurate, fast and reliable basis for his choice of therapeuticagent.

Traditional anti-microbial sensitivity assay techniques are routinelyaccomplished by obtaining a patient sample containing the infectiousagent and adding the sample to a series of test tubes containing agrowth media. In all but one of the test tubes (a control), are addeddifferent antibiotics (one per tube) which are potentially useful fortreatment of the individual affected with the infectious agent.Alternatively, sensitivity testing may be performed by using a petridish containing growth media onto which the bacterium being tested isstreaked. Following the placement of a series of impregnatedantibiotic-containing "disc's" onto the surface of this medium, the drugof choice is determined by measuring the amount of growth after apredetermined incubation period. As with the test tube method, theregion about the antibiotic showing the greatest degree of clearing(i.e., demonstrating the least amount of bacterial growth) is indicativeof which drug has best inhibited the growth of the microbe. This drug isthus the drug of choice in treatment of the microbial infection. Themethod of measuring the degree of growth of the infectious agent in apatient sample in the presence of different drugs is the subject of thisinvention.

(c,d) A technique currently used for microbial antigen detection andidentification is that of ELISA, or enzyme-linked immunosorbent assay.In a classical heterogeneous assay (such as described in U.S. Pat. No.3,654,090), microbial identification is performed in a series ofmicrotiter wells coated with antibody. This type of assay generallyinvolves at least two incubation periods and several wash steps. Here,too, there are numerous limitations associated with the system. As withthe microoganism identification systems discussed above, two incubationperiods are required. Many of the other limitations and deficienciesassociated with the microorganism identification systems discussedhereinabove are also present in microbial identification by an ELISAtechnique. Moreover, the typical microtiter plate consists of ninety-six(96) wells. If the number of assays to be performed is less than thetotal number of wells, the cost per test is increased proportionally.

The problems and shortcomings associated with the techniques describedhereinabove are encountered to a similar extent in microbialidentification assays, the determination/quantitation of viral surfaceantigen, and the corresponding detection of serum antibodies againstthese microbial antigens.

As is evident from the above discussion, there is a continuing need forimprovement in the currently available techniques for surface antigendetection in the following respects: reduction in the number ofmanipulative steps which are performed manually by the tehnician;reduction in the time required for the performance of such assays;enhancement in the accuracy of the reliability of the test results; and,reduction in the cost per test charge to the patient.

OBJECTS OF THE INVENTION

Accordingly, it is the object of this invention is to remedy the aboveas well as related deficiencies in the prior art.

More specifically, it is the principal object of this invention toprovide an automated method for the detection of infectious agents influid samples.

It is another object of this invention to provide a method for theevaluation of a patient clinical sample for identification of aninfecting microorganism.

It is yet a further object of this invention to provide a method for thedetermination of an efficacious antibiotic by performing ananti-microbial sensitivity assay.

It is still yet a further object of this invention to provide a methodfor the semi-quantitative determination of viral surface antigen.

It is still yet a further object of this invention to provide a methodfor the identification and quantitative determination of serumantibodies to microbial antigens.

SUMMARY OF THE INVENTION

The above and related objects are achieved by providing a method fordetection of microbial antigen or serum antibody to such antigen(hereafter "analyte") by radial partition immunoassay. As isappreciated, the identification of a microorganism can also be achievedindirectly through identification of characteristic products of theirmetabolism and/or component parts thereof resulting from thefragmentation, lysing and/or cleavage of the microorganism. The term"analyte" as used throughout this disclosure is intended as not onlydescriptive of infectious agents (i.e. microbes) of the type which caninduce disease within a host organism and which generally proteinaciousin composition or of a host-produced antibody against such an organismbut also other products and/or component parts thereof which areindicative of the presence of the microorganism. The assay of a fluidsample in accordance with this method is conducted entirely within asolid, inert immobilizing porous matrix by initially applying a fluidsample containing an unknown quantity of analyte to a finite zone of theimmobilizing porous matrix. The analyte is applied either as a solutionor a suspension so as to permit dispersion thereof within theinterstices of such matrix. The distribution of analyte within suchmatrix defines the boundaries of a reaction zone within this matrix.Subsequently, a labeled compound or indicator is applied as a liquid tosubstantially the center of the reaction zone, under conditions whichallow the indicator to immunochemically bind to the analyte in an amountwhich can be correlated to the amount of analyte in the reaction zone.The labeled compound or indicator is usually a compound moleculeconsisting of a binding agent having an affinity for the analyte ofinterest chemically conjugated to the signal generating moiety.Following a brief incubative period, a stream of eluting solvent isapplied to substantially the center of the reaction zone, the quantityof this eluting solvent being sufficient to effect a radialchromatographic separation, entirely within the immobilizing porousmatrix, of unbound indicator and endogeneous materials from theindicator which is bound within the reaction zone. After such separationis effected, the extent to which the bound indicator is present within adelimited area of the reaction zone is observed by measurement withappropriate instrumentation. As a result of the foregoing separationstep (radially partitioning), the delimited area of the reaction zone isessentially free of unbound indicator.

The foregoing method is applicable to an anti-microbial sensitivityassay, a microbial identification assay, the detection of microbialantigens, and the detection of antibodies to microbial and viralmicrobial antigens.

DESCRIPTION OF THE INVENTION INCLUDING THE PREFERRED EMBODIMENTS

The method of this invention, as applied to an anti-microbialsensitivity assay, initially involves the culturing of microbes obtainedfrom fluid samples in vitro in a growth media containing one or morepotentially effective antibiotics. The various antibiotics present ineach of the cultures inhibits the growth of the microbe to a greater orlesser degree, and, thus, the test tube containing the least microbialgrowth is indicative of the most efficacious drug or drug combination,for the treatment of the microbial infection in the afflicted patient.

For the purposes of the description of this portion of the invention,the terms "analyte" and "microbe" are used interchangeably andcontemplate microoganisms not only of bacterial size, but is alsoinclusive of fungi, Rickettsia, protozoa, viruses or any othermimcroorganism, which can be effectively entrapped in a prepared porousmatrix of the type typically used in the filtration of bacterialcultures, i.e., Whatman Glass Microfilter Filters.

In practice, a sample of inoculum is added to each of a series of testtubes containing a growth media and, in all but one test tube, differentantibiotics are added, one per test tube. The contents of each tube isthereafter suspended and subsequently incubated. An aliquot of thissuspension can then be applied to a porous matrix wherein the microbebecomes entrapped and immobilized with the matix over an area whichcorresponds to distribution of the fluid sample therein. Thedistribution of the fluid within the porous matrix generally defines theboundaries of a reaction zone within which the screening method of thisinvention can be performed. Since direct measurement of theconcentration of microbe is difficult, the method of this inventioninvolves the measurement of a labeled compound, whose concentrationwithin the matrix can be correlated to the concentration to microbe ofinterest.

Assuming, for illustration purposes, that the immobilized microbe hascharacteristic surface antigens, its identification by immunochemicalinteraction with appropriately labeled antibody to such surfaceantigen(s) can be readily accomplished in accordance with radialpartition immunoassay methodology, as adapted herein to theanti-microbial sensitivity assay; a comprehensive description of thetechnique applicable to the assays of this invention appearing in,Giegel et al, Radial Partition Immunoassay, Clin. Chem., 28:9, 1894(1982); copending U.S. patent application Ser. No. 227,664, filed Jan.23, 1981; and Spanish Patent No. 508,875 (to Giegel and Brotherton)-allof which are hereby incorporated by reference in their entirety.Following the immobilization of the microbe in the porous matrix in themanner described hereinabove, a labeled antibody is subsequently appliedto the reaction zone of the porous matrix containing the sample underconditions which favor immunochemical binding of the labeled antibodywith the surface antigen of the microbe entrapped in the matrix. After abrief incubation period, a stream of wash fluid is applied to the porousmatrix to effect separtion of unbound materials from that portion of thereaction which is to be monitored for the presence of the microorganismof interest. In the most preferred embodiments of this invention thepoint of application of the aliquot of patient's sample, labeledantibody and wash fluid is essential coincident. However, so long asthere is substantial overlap in the migration patterns of the fluidsample, labeled antibody and wash fluid, precise coincidence ofapplication to the porous matrix is not required.

These unbound materials generally migrate radially from the point ofapplication of wash fluid. Depending upon the type of label used, thelevel of bound labeled antibody can be monitored by measurement with thetype of fluorometric or spectrophotometric devices disclosed in U.S.Pat. No. 4,059,405 (which is hereby incorporated by reference in itsentirety) or by counting the amount of radioactivity in the reactionzone. The level of labeled antibody which is present in the sample isthereafter compared to a standard or control. This comparison can alsobe made manually (i.e., visually) or, where the monitoring of indicatorlevel is automated, through the use of microprocessors. In theinterpretation of the results of such assay, the control samplecontaining no antibiotic will have the highest level of labeled antibodyindicating the greatest amount of growth. The samples which have beeninnoculated with antibodies, will have a lower level of labeledantibody, indicating inhibited growth due to the presence and relativeeffectiveness of the antibiotic.

As noted above, the method of this invention is performed within aninert porous matrix. The porosity of the matrix is sufficiently fine toentrap particles of microbial size thereby effectively immobilizing themwithin its interstices. The entrapment of the microbes within theinterstices of the matrix allows for the matrix to act as a uniquereaction vessel for immunochemical interactions between the analyte andthe indicator. The matrix is selected so that the size of theinterstitial spaces is sufficiently small to effectively immobilizemicrobial size particles yet allow the reaction fluid to spread, viacapillary action, essentially uniformally in all directions. When analiquot of patient sample is dispensed onto the matrix, the microbe willbe principally immobilized at and peripheral to the point of applicationthus, defining the reaction zone for subsequent interaction with otherreagents (i.e. labeled indicator). The relatively large surface areaassociated with a porous matrix of this nature permits the concentrationof the analyte within a comparatively small area, thereby decreasing theamount of patient sample necessary to obtain satisfactory results andincreasing the overall sensitivity and efficiency of the method.

This porous matrix can be composed of any material which is inert, i.e.,that is will not itself react deleteriously with any materials that arebrought in contact with it which are associated with the particularassay. In addition, in the context of this invention, "inertness"contemplates that the matrix will not chemically or immunologicallyinteract with either the sample or the reagents used in performance ofthe assay, will not dissolve and has negligible nonspecific attractionfor such materials. In those embodiments of this invention where thephysical size of the analyte of interest is too small for effectivephysical entrapment, and therefore immobilizing the analyte within theinert matrix, the matrix can be sensitized with an appropriate agent(i.e., antibody) to bind such finer particles. As noted above, one ofthe principal advantges of the use of a porous matrix is its largesurface area which enables concentration of the analyte of interest in asmall area. In the preferred embodiments of this invention, the porousmatrix is advantageously made of a mat of composed fibers, such as glassor synthetic fibers. The matrix may, however, by composed of othernon-fibrous porous materials such as sintered glass, ceramics, syntheticspongy materials or polymers, etc. Since one of the essential featuresof the radial partition immunoassay methodology is the ability of thistechnique to effect rapid and complete separation of constituents ofdiffering mobility within the matrix, the presence of anything withinthe matrix which would be incompatible with or inhibit the capillaryflow of liquids in such matrix is obviously unsatisfactory. Thus,gelatinous solids and the like are, by definition, excluded from thecategory of matrices which are accpetable for use in this method. Othermaterials such as cellulosic paper tend to have a greater nonspecificattraction for the material applied thereto and thus may requirepretreatment to inactivate their reactive sites.

The method of this invention is useful for a variety of biologicalassays. For example, a throat swab, blood or urine sample can be quicklyand accurately analyzed for various bacterial and other microbes.Microorganism analytes of interest include: Corynebacteria, Pneumococci,Streptococci, Staphylococci and Neisseria. Some of the above mentionedmicroorganisms may need to be pretreated by fragmentation, lysing,cleaving, etc., in order to obtain an appropriate fraction or portionsuitable for analysis. Viral assay which may be performed in accordancewith the methodology of this invention includes: Adenoviruses, HerpesViruses, Pox Viruses, Picornaviruses, Myxoviruses, Arboviruses,Reoviruses and Tumor Viruses. Hepatitis A and B as well as other formsof hepatitis can be detected by testing for the presence of hepatitisviral antigen or antibody in the patient sample. To entrap particles ofsmaller size within the matrix, (such as the viruses mentioned above),the matrix may have to be pretreated with an antibody or other agent.Virtually any type of biological fluid can be effectively screened inaccordance with above methodology for the presence infectious agents.

As is typically done, a throat swab can be used for a bacterial screenof sputum sample to detect Staphylococcus aureus, Haemophilusinfluenzae, Streptococcus pneumoniae, Psudomonas aeruginosa,Enterobacteriaceae (group), Neisseria meningitidis, Corynebacteriumdiptheriae, Streptococcus pyogenes and Bordetella pertussis.

Similary, a urine screen can be utilized for detection of Escherichiacoli, Proteus mirabilis, Pseudomonas aeruginosa or Staphylococcus Sp.Also, infectious agents which appear in blood, serum or spinal fluid canbe detected using the method of this invention.

Standard dilutions of the sample in liquid medium are typically preparedso that a uniform aliquot can be added to the porous matrix.

After the analyte of interest is immobilized within the matrix, alabeled compound is introduced into the reaction zone containing theinfectious agent. The purpose of the label is to provide an indirectmeans for measuring the amount of analyte present in the sample. Thelabeled compound is designed to immunochemically bind to the analyteentrapped with the matrix. A stream of eluting solvent (wash fluid) canthen be applied to effect radial partitioning of unbound labeledcompound and other unbound materials from that portion of the reactionzone which is to be monitored for the presence of analyte. Afterapplication of the wash fluid in the foregoing manner, only that portionof the labeled compound that has bound to the analyte which is entrapped(within the matrix) is detectable for subsequent measurement by themonitoring techniques. In an alternate embodiment of the presentinvention, the sample solution and solution containing the labelledcompound are pre-mixed prior to application to the porous medium.

The labeled compounds suitable for use in this method can be anantibody, antigen or any other substance that selectivelyimmunochemically binds with the analyte of interest and will eitheritself produce a detectable signal or interact with yet another reagentto produce a detectable signal. The synthesis of labeled compoundssuitable for use in the method of this invention can be performed bywell-known synthetic protocols. Typically, such synthesis involvescross-coupling, or conjugation, of an indicator molecule to animmunochemically active fragment through a bifunctional reagent. Thus,the term "conjugate" or phrase "labeled conjugate" is also used in theart throughout this disclosure interchangeable with the term "indicator"and phrase "labeled compound" respectively. Various indicators which aresuitable for use in the methodology of this invention includeradiolabels such as ¹²⁵ I or tritium, fluorescent molecules, enzymes,cofactors, chemiluminscence reagents and compounds that becomefluorscent upon subsequent enzymatic attack. In the case of radioactiveindicators the concentration of the labeled compound is usuallymonitored by placing the porous medium having the bound labeled compoundin a scintillation or gamma counter. The extent of enzyme labeledcompound bound to the analyte within the reaction zone can be detectedby simply contacting the bound enzyme labeled compound with a substrate,under conditions which favor enzymatic cleavage of an indicator moleculefrom substrate. The indicator molecule is preferably a chromogenic orfluorogenic substance that can be measured by conventional monitoringtechniques. Compounds labeled with enzyme cofactors or effectors can bedetected similarly by their effect on enzyme action on a substrate.Compounds labeled with chromophores may be directly measureable usingfluoroscopy, ultraviolet spectroscopy or other spectroscopic or visualmeans.

After the labeled compound has been added to the reaction zone, in themanner described previously, a brief incubation period is generallyrequired to permit immunochemical interaction of the labeled compoundand the analyte. A clean separation of unbound label compounds, as wellas interfering endogenous proteins, must then be effected from adelimited area of the reaction zone prior to the monitoring step. Thissepartion, or partitioning, is accomplished by applying a stream of aneluting solvent to the reaction zone in sufficient volume to effectradially chromatographic separation of unbound materials from thereaction zone. As noted previously, so long as there is substantialoverlap in the patterns of distribution of the various fluids which areapplied to the porous matrix, an effective separtion, or partitioning ofbound and free labeled compound will occur. The solvent employed in thiselution step may be water or a buffer solution in which such unboundcompounds are conveniently dissolved. As the solvent migrates radiallyout from its point of application to the reaction zone, unboundreactants are separated from the bound reactants. A small quantity ofsuch solvent is generally sufficient to cleanly separate the unboundreactants from at least a portion of the reaction zone. Such reactants,if visible, generally appear as one of more rings around the reactionzone, with the distance of separation only being dependent on the volumeof the solvent used and the Rf values for the reactants. Small volumesof eluting solvent have been found effective for achieving goodseparation of free from bound reactants, thus providing a quick,economical and reliable assay procedure. Typically, solvent volumes offrom about 10 microliters to about 150 microliters, and preferably 50 to80 microliters are employed. These solvents may be conveniently appliedto the reaction zone with a pipette, hypodermic syringe of otherdispensing device. The volumes of fluids applied to the porous matrix inthe performance of the assay for this invention are sufficient to effectan essentially homogeneous area within the reaction zone for monitoringof the analyte of interest; and, yet permit retention of all of suchfluids entirely within the porous matrix containing the immobilizedanalyte.

Upon completion of the immunochemical reaction and the partitioning ofthe bound and unbound labeled compounds within the reaction zone, adelimited area of this zone is monitored, with the aid of appropriateinstruments, to determine the magnitude of the signal generated by thelabeled compound. The delimited area of reaction zone (generally 6-10 mmin diameter) is confined to only a portion of the matrix within whichthe entrapped microbes and bound labeled compound are present. Thisdelimited area is predetermined by simple adjustment in the aperatureassociated with the means for detection of the level of chromophore,fluorophore or radiolabel.

The signal produced by the labeled compound, or the action of thelabeled compound on a appropriate substrate, can be routinely monitoredwith responsive instrumentation. The type of instrumentation employedwill, of course, depend upon the type of immunoassay performed and thelabel used. Instrumentation which is suitable for use in conjunctionwith the method of this invention is described in U.S. Pat. No.4,059,405, issued Nov. 22, 1977; commonly assigned copending U.S.application of Heller, Ser. No. 401,670 filed July 26, 1982; and, inGiegel et al, "Radial Partition Immunoassay", Clinical Chemistry, 28:9,1982; all of the above being hereby incorporated by reference in theirentirely. Typically, colorimetric, ultraviolet or fluorescent assays maybe used for rate determinations (kinetic assays). The rate of formation(or disappearance) of the measured chromophore or fluorophore istypically compared to a calibration standard as an indication ofconcentration of the analyte. Such measurements may be made directlyfrom the surface of the porous medium, employing a front surfacefluorometer or reflectometer. The method of this invention is fullycompatible with automated systems and provides an accurate quantitationof the amount of analyte present in the sample. Alternatively, thismethod may also be used as a qualitative or semiquantitative test todetermine the presence, absence or approximate indication of the levelof infectious agent.

The method of this invention is also applicable to the analysis of apatient clinical sample for identification of a microorganism (orantibodies against that microorganism). One common method for thequantitation of antibodies in patient sample is the sandwich assay. Theprocedure for performance of various sandwich assays which are describedin U.S. Pat. No. 4,376,110 ( David et al) are also fully compatible withthe radial partition immunoassay methodology of this invention, theDavid et al patent being incorporated by reference in its entirety.

A sandwich assay generally involves the binding of analyte of interestfrom a patient sample to either an antigen or antibody here after"ligand") which has been pre-immobilized on a porous matrix. Thedistribution of analyte, which is bound to the immobilized liganddefines the parameters of the reaction zone within the matrix. In one ofthe preferred embodiments for the detection of analytes too small to beentrapped within the matrix, the amount of immobilized ligand(sensitizing agent) within the matrix is in excess to insure essentiallycomplete binding of the analyte of interest. Sufficient incubationperiod is allowed to elapse to permit immunochemical interaction of theanalyte with the ligand. A labeled ligand such as a labeled antibody isthereafter applied to the reaction zone of the matrix and also allowedto interact with the analyte from the patient sample. After a secondbrief incubation period a wash fluid is applied to the matrix to effectradial elution of substantially all unbound labeled ligand (antibody)from at least a delimited area of the reaction zone. The signalgenerated by the labeled ligand can then be monitored in the mannerpreviously described. As with the anti-microbial sensitivity assaydescribed hereinabove, any of the traditional type of indicator, e.g.,fluorescent, enzyme, cofactor, radioactive, chromatogenic, etc., can beused to label the ligand. After the labeled ligand is applied to thereaction zone and allowed to react, an eluting solvent is employed toremove any unbound material from this zone. Depending upon theparticular indicator compund that is used, the eluting solvent may alsocontain a substrate, which, upon interaction with the bound labeledligand, releases a detectable signal that can be measured and correlatedto the concentration of analyte in the reaction zone. A delimited areaof the reaction zone is monitored for the presence of the indicator.

The method of this invention is also applicable to the semi-quantitativedetermination of viral surface antigens or antibodies, such as HepatitisA and B and Rubella viruses via a sandwich assay or via the othersimilar assays herinabove described.

Another of the significant advantages of this methodology, is itscompatability with automated instrumentation (i.e. STRATUS ImmunoassaySystem) of the type which is commercially available from American DadeCompany, American Hospital Supply Corporation, this system being morefully described by Giegel et at, in an article relating to radialpartition immunoassay referenced hereinabove. In this automated device,the porous matrix is retained in a self-stacking tab similar inconfiguration to photographic transparency, and the sample, labeledindicator and wash solution added thereto by a series of automatedpipettes in the appropriate sequence. The construction of theself-stacking tab, which is compatible with the STRATUS ImmunoassaySystem, is more fully described in U.S. Pat. No. 4,440,301 to FranklinS. Intengan, (which is hereby incorporated by reference in itsentirety). The measurement of the amount of labeled indicator bound inthe matrix is performed by front-surface fluorescence, and the entireprocedure is microprocessor controlled. This system has the capabilityto perform thirty-two of the more common immunoassays which arepresently performed in a clinical laboratory.

EXAMPLES

The Examples which follow further describe, define and illustrate anumber of different embodiments of this invention. Parts and percentagesappearing in such examples are by weight unless otherwise stipulated.The apparatus and techniques used in the preparation of reagents and/orperformance or evaluation of the method of this invention are standardof as herein before described. All of the following assays are performedon a STRATUS automated immunoassay system utilizing the radial partitionimmunoassay methodology developed for this system. The instrument ismodified as appropriate depending upon the choice of indicator.

I. IDENTIFICATION OF SPECIFIC MICROBES (OR MICROBIAL ANTIGENS) Example1: Detection of Haemophilus influenzae

1. A 50 ul sample of cerebral spinal fluid (CSF) is spotted onto thesurface of a Stratus reagent tab containing preimmobilized Anti-H.influenzae antibody. The reagent tabs to be used with this example areprepared by premixing rabbit anti-H. influenzae antibody with goat antirabbit antibody followed by spotting onto the surface of Whatman GF/Fglass fiber filter paper.

2. After a 5 minute incubation, the tabs is then spotted with 50 ul ofalkaline phosphatase labeled mouse anti-H. influenzae.

3. After an additional 5 minute incubation, unbound alkaline phosphataselabeled mouse anti-H influenzae is washed from the central field of viewby the addition of a substrate wash solution.

4. Quantitation is performed via Stratus methodology. Appropriatepositive and negative controls are also assayed using different tabs inthe same run.

Example 2: Detection of Neisseria meningitidis

1. A 50 ul sample of cerebral spinal fluid (CSF) is spotted onto thesurface of a Stratus reagent tab containing preimmobilized Anti-N.meningitidis antibody. The reagent tab to be used in this example isprepared by premixing rabbit anti-N. meningitidis antibody with goatanti rabbit antibody followed by spotting onto the surface of WhatmanGF/F glass fiber filter paper.

2. After a 5 minute incubation, the tabs is then spotted with 50 ul ofalkaline phosphatase labeled mouse anti-N. meningitidis.

3. After an additional 5 minute incubation, unbound alkaline phosphataselabeled mouse anti-N. meningitidis is washed from the central field ofview by the addition of a substrate wash solution.

4. Quantitation is performed via Stratus methodology. Appropriateposition and negative controls ae also assayed using different tabs inthe same run.

Example 3: Detection of Staphylococcus aureus

1. A throat swab containing the clinical sample is placed into a smallvolume of culture broth.

2. A 50 ul sample of the suspension is then spotted onto the surface ofa Stratus reagent tab containing preimmobilized Anti-S. aureus antibody.The tab to be used in this Example is prepared by premixing rabbitanti-S. aureus antibody with goat anti rabbit antibody followed byspotting onto the surface of Whatman GF/F glass fiber filter paper.

3. After a 5 minute incubation, the tabs is then spotted with 50 ul ofalkaline phosphatase labeled mouse anti-S. aureus.

4. After an additional 5 minute incubation, unbound alkaline phosphataselabeled mouse anti-S. aureus is washed from the central field of view bythe addition of a substrate wash solution.

5. Quantitation is performed via Stratus methodology. Appropriatepositive and negative controls are also assayed using different tabs inthe same run.

Examples 4-6

The procedures of Examples 1-3 are repeated except that the clinicalsamples are serum.

Examples 7-9

The procedures of Examples 1-3 are repeated except that the clinicalsamples are urine.

Examples 10-18

The procedures of Examples 1-9 are repeated except that "blank" tabsrather than those containing preimmobilized anti-microbe antibody areused in step 1. The GF/F paper has a reported average pore size of 0.7microns and physically entrap the analyte within the matrix.

Example 19: Detection of Hepatitus B Antigen

1. A 50 ul serum sample is spotted onto the surface of a Stratus reagenttab containing preimmobilized Anti-Hepatitus antibody. The reagent tabto be used in this example is prepared by premixing rabbitanti-Hepatitus antibody with goat anti rabbit antibody followed byspotting onto the surface of Whatman GF/F glass fiber filter paper.

2. After a 5 minute incubation, the tabs is then spotted with 50 ul ofalkaline phosphatase labeled mouse anti-Hepatitus.

3. After an additional 5 minute incubation, unbound alkaline phosphataselabeled mouse anti-Hepatitus is washed from the central field of view bythe addition of a substrate wash solution.

4. Quantitation is performed via Stratus methodology. Appropriatepositive and negative controls are also assayed using different tabs inthe same run.

Example 20: Detection of Rubella Antigen

1. A 50 ul serum sample is spotted onto the surface of a Stratus tabcontaining preimmobilized Anti-Rubella antibody. The reagent tab to beused in this Example is prepared by premixing rabbit anti-Rubellaantibody with goat anti rabbit antibody followed by spotting onto thesurface of Whatman GF/F glass fiber filter paper.

2. After a 5 minute incubation, the tabs is then spotted with 50 ul ofalkaline phosphatase labeled mouse anti-Rubella.

3. After an additional 5 minute incubation, unbound alkaline phosphataselabeled mouse anti-Rubella is washed from the central field of view bythe addition of a substrate wash solution.

4. Quantitation is performed via Stratus methodology. Appropriatepositive and negative controls are also assayed using different tabs inthe same run.

Examples 21-40

The procedures of Examples 1-20 are repeated except that a fluorophore(fluorescein) is used as the label in place of the alkaline phosphatase.

Examples 41-80

The procedures of Examples 1-40 are repeated except that the assays arerun in a competitive mode using alkaline phosphatase labeled antigen inplace of the respective alkaline phosphatase labeled antibodies.

Examples 81-91

The procedures of Examples 1-9, 19 & 20 are repeated except that theantibody is immobilized onto the surface of the Stratus reagent tab viachemical crosslinking reagents such as glutaraldehyde.

II. IDENTIFICATION OF SPECIFIC ANTIBODIES TO MICROBES Example 92:Detection of Serum IgM Antibodies Against Rubella.

1. A 50 ul serum sample is spotted onto the surface of a Stratus reagenttab containing preimmobilized Rubella antigen. The reagent tab to beused in this Example is prepared by premixing a complex of rabbitanti-Rubella antibody and Rubella antigen with goat anti-rabbit IgGfollowed by spotting onto the surface of Whatman GF/F glass fiber filterpaper.

2. After a 5 minute incubation, the tabs is then spotted with 50 ul ofan alkaline phosphatase labeled rabbit anti-human IgM solution.

3. After an additional 5 minute incubation, unbound alkaline phosphataselabeled rabbit anti-human IgM was washed from the central field of viewby the addition of a substrate wash solution.

4. Quantitation is performed via Stratus methodology. Appropriatepositive and negative controls are also assayed using different tabs inthe same run.

Example 93: Identification of IgG Antibodies to Hepatitis Antigen.

1. A 50 ul serum sample is spotted onto the surface of a Stratus reagenttab containing preimmobilized Hepatitis antigen. The reagent tab to beused in this Example is prepared by premixing a complex of rabbitanti-Hepatitis and Hepatitis antigen with goat anti-rabbit IgG followedby spotting onto the surface of Whatman GF/F glass fiber filter paper.

2. After a 5 minute incubation, the tabs is then spotted with 50 ul ofan alkaline phosphatase labeled rabbit anti-human IgG solution.

3. After an additional 5 minute incubation, unbound alkaline phosphataselabeled rabbit anti-human IgG is washed from the central field of viewby the addition of a substrate wash solution.

4. Quatitation is performed via Stratus methodology. Appropriatepositive and negative controls are also assayed using different tabs inthe same run.

Example 94: Detection of IgG Antibodies to Neisseria gonorrhoeae

1. A 50 ul serum sample is spotted onto the surface of a Stratus reagenttab containing preimmobilized N. gonorrhoeae antigen. The tab to be usedin this Example is prepared by premixing a complex of rabbit anti-N.gonorrhoeae antibody and N. gonorrhoeae antigen with goat anti-rabbitIgG followed by spotting onto the surface of Whatman GF/F glass fiberfilter paper.

2. After a 5 minute incubation, the tabs is then spotted with 50 ul ofan alkaline phosphatase labeled rabbit anti-human IgG solution.

3. After an additional 5 minute incubation, unbound alkaline phosphataselabeled rabbit anti-human IgG is washed from the central field of viewby the addition of a substrate wash solution.

4. Quantitation is performed via Stratus methodology. Appropriatepositive and negative controls are also assayed using different tabs inthe same run.

Examples 95-97

The procedures of Examples 92-94 are repeated except that a fluorophore(fluorescein) is used as the label in place of the alkaline phosphatase.

Examples 98-103

The procedures of Examples 92-97 are repeated except that the antigen isimmobilized onto the surface of the Stratus reagent tab via chemical,rather than by immunological means.

III. MICROBIAL SCREEN Examples 104: Identification of Staphylococcusaureus from an "Unknown" Clinical Specimen

1. A throat swab containing the clinical sample is placed into a smallvolume of sterile saline.

2. A 50 ul sample of the suspension is then spotted onto the surface ofa Stratus reagent tab containing preimmobilized Anti-S. aureus antibody.The reagent tab to be used in this Example is prepared by premixingrabbit anti-S. aureus antibody with goat anti rabbit antibody followedby spotting onto the surface of Whatman GF/F glass fiber filter paper.

3. After a 5 minute incubation, the tab is then spotted with 50 ul ofalkaline phosphatase labeled mouse anti-S. aureus.

4. After an additional 5 minute incubation, unbound alkaline phosphataselabeled mouse anti-S. aureus is washed from the central field of view bythe addition of a substrate wash solution.

5. Quantitation is performed via Stratus methodology. Appropriatepositive and negative controls are also assayed using different tabs inthe same run.

Examples 104-113

The procedure of Example 104 except that the microbes and rabbitantibodies against those microbes) are as follows:

a. Haemophilus influenzae.

b. Streptococcus pneumoniae.

c. Pseudomonas aeruginosa.

d. Enterobacteriaceae (group).

e. Neisseria meningitidus.

f. Corynebacterium diphtheriae.

g. Streptococcus pyogenes.

h. Bordetella pertussis.

Examples 114-123

The procedures of Examples 104-113 are repeated except that the label isa fluorophore (fluorescein) rather than alkaline phosphatase.

Examples 124-143

The procedures of Examples 104-123 are repeated except that "blank"glass fiber filter paper tabs are used (without previously immobilizedantibody present).

Examples 144-163

The procedures of Examples 104-123 are repeated except that theimmobilization of the antibodies within the tab is via a chemicalcrosslinking agent such as glutaldehyde.

IV. ANTIMICROBIAL SENSITIVITY TESTING Example 164

1. An aliquot from a standardized inoculum obtained from a clinicalsample is placed into a series of tubes containing a suitable growthmedium.

2. Into each tube (with the exception of one, the "control") is placed adifferent antimicrobial agent that might be effective against thatparticular microbe.

3. The tubes are then incubated for a period of at least 1-2 hours inorder to promote growth of the microorgnism.

4. An aliquot from each of the tubes is then spotted onto individual"blank" glass fiber filter paper tabs.

5. An aliquot containing a mixture of alkaline phosphatase-labeledantibodies (against the various broad groupings of microbial antigens)is then applied to the tabs.

6. The tabs are incubated for 5 minutes.

7. Addition of substrate wash solution follows the brief incubationstep. Quantitation is performed via the Stratus instrument.

8. All assay valves are compared to that of the control. Decreasedenzymatic activity to that of the control indicates that thatantimicrobial agent is somewhat effective in inhibiting growth.

Example 165

The procedure of Example 164 is repeated except that the fluorophorefluorescein replaces the alkaline phosphatase as the label.

Example 166

The procedure of Example 164 is repeated except that the tabs contain anagent that sides in the immobilization of the microbial cells (i.e., thetabs are NOT "blank").

Examples 167-168

The procedures of Examples 164 & 166 are repeated except that thelabeled antibody conjugate is omitted. The substrate in the washsolution is matched to specificity of the immobilized microorganism. Theaction of the microorganism on the substrate releases the fluorophore.

Examples 169-170

The procedures of Examples 164 and 166 are repeated except that theimmobilized microbial cells are "lysed" by a "substrate" reagent thatalso contains the materials necessary to assay for a specific componentreleased from within these microbial cells (i.e., NAD, a specificenzyme, a specific metabolite, etc.).

What is claimed is:
 1. A solid phase radial partition immunoassay fordetection of microbial analyte in fluid samples, said immunoassaycomprising:(a) applying an aliquot of a fluid sample, containing anunknown level of microbial analyte, to a finite reaction zone of a solidinert porous medium so as to effectively immobilize said analyte withinthe reaction zone of the porous medium; (b) applying a labeled compoundconsisting essentially of an indicator conjugated to an antibody, tosubstantially the center of said reaction zone under conditions whichfavor the immunochemical interaction of the analyte with the antibody ofthe labeled compound within a delimited area of said reaction zone, suchinteraction of said labeled compound being in proportion to the amountof analyte in said delimited area; (c) applying a wash solution,comprising an eluting solvent, to substantially the center of thereaction zone in a quantity sufficient to effect radial chromatographicseparation, within said porous medium, of the unbound labeled compoundfrom that which is bound to the immobilized analyte within the delimitedarea of the reaction zone; and (d) observing the extent to which thebound labeled compound is present within the delimited area of saidreaction zone by measurement of the level of indicator in said delimitedarea.
 2. The assay of claim 1, wherein the porous medium consistsessentially of a filter paper and said microbial analyte is physicallyentrapped within the interstices of said paper.
 3. The assay of claim 1,wherein the porous medium is presensitized with a ligand specific for animmunochemical binding site on said microbial analyte, so as to rendersaid medium retentive of said microbial analyte.
 4. The assay of claim1, wherein said microbial analyte is selected from a group consisting offungi, Rickettsia, protozoa, and viruses.
 5. The assay of claim 1,wherein said microbial analyte consists essentially of proteinaceousconstituents of the fragmentation, lysing or cleavage of amicroorganism.
 6. The assay of claim 1, wherein the indicator of thelabeled compound is selected from the group consisting of enzymes,fluorophores, chromophores and radioactive isotopes.
 7. The assay ofclaim 1, wherein the indicator of the labeled compound is an enzyme andthe wash solution contains a substrate for said enzyme, wherein theenzymatic action of said enzyme on said substrate results in cleavage ofan indicator from said substrate.
 8. The assay of claim 1, wherein saidmicrobial analyte is Hepatitis virus.
 9. The assay of claim 1, whereinsaid microbial analyte is Herpes virus.
 10. The assay of claim 1,wherein said microbial analyte is a species of bacteria.
 11. A solidphase radial partition immunoassay for detection of a microbial analytein fluid samples, said microbial analyte consisting essentially of asoluble antigen which has been secreted by a microorganism, or afragment of the microorganism, the immunoassay comprising the stepsof:(a) providing a solid, inert porous medium that has been pretreatedwith an immobilization effective amount of an antibody which is specificfor an immunochemical binding site on said microbial analyte so as torender said medium retentive of said microbial analyte; (b) applying analiquot of a fluid sample, containing an unknown level of microbialanalyte, to a finite reaction zone of a solid, inert medium so as toeffectively immobilize said analyte within the reaction zone of theporous medium; (c) applying a labeled compound, consisting essentiallyof an indicator conjugated to an antibody, to substantially the centerof said reaction zone under conditions which favor the immunochemimcalinteraction of the analyte with the antibody of the labeled compoundwithin a delimited area of said reaction zone, such interaction of saidlabeled compound being in proportion to the amount of analyte in saiddelimited area; (d) applying a wash solution, comprising an elutingsolvent, to substantially the center of the reaction zone in a quantitysufficient to effect radial chromatographic separation, within saidporous medium, of the unbound labeled compound from that which is boundto the immobilized analyte within the delimited area of the reactionzone; and (e) observing the extent to which the bound labeled compoundis present within the delimited area of said reaction zone bymeasurement of the level of indicator in said delimited area.
 12. Theassay of claim 11, wherein the microbial analyte is a virus.
 13. Theassay of claim 11, wherein the sample solution and solution containingthe labeled compound are pre-mixed prior to application thereof to theporous medium.
 14. The assay of claim 13, wherein the microbial analyteis the hepatitis B virus, and the porous medium is pretreated withanti-serum to Hepatitis B surface antigen.
 15. The assay of claim 11,wherein the microbial analyte is a viral fragment.
 16. A solid-phaseradial partition immunoassay for detection of serum antibody to amicrobial antigen in a fluid sample, the immunoassay comprising thesteps of(a) providing a solid inert porous medium that has beenpretreated with an immobilization effective amount of antigen which isspecific for an immunochemical binding site on the serum antibody so asto render said medium retentive of said antibody; (b) applying analiquot of a fluid sample, containing an unknown level of said serumantibody, to a finite reaction zone of said solid inert porous medium soas to effectively immobilize said serum antibody within the reactionzone of said porous medium; (c) applying a labeled compound, consistingessentially of an indicator conjugated to an anti-antibody tosubstantially the center of said reaction zone under conditions whichfavor the immunochemical interaction of the antibody with theanti-antibody of the labeled compound within a delimited area of saidreaction zone, such interaction of said labeled compound being inproportion to the amount of antibody in said delimited area; (d)applying a wash solution, comprising an eluting solvent, tosubstantially the center of the reaction zone in a quantity sufficientto effect radial chromatographic separation, within said porous medium,of the unbound labeled compound from the immobilized antibody within thedelimited area of the reaction zone; and (e) observing the extent towhich the bound labeled compound is present within the delimited area ofsaid reaction zone by measurement of the level of indicator in saiddelimited area.
 17. A solid-phase radial immunoassay for detection of amicrobial analyte in fluid samples, said immunoassay comprising:(a)providing a solid inert porous medium that has been pretreated with animmobilization effective amount of an antibody which is specific for animmunochemical binding site on said microbial analyte so as to rendersaid medium retentive of said microbial analyte; (b) applying an aliquotof a fluid sample, containing an unknown level of microbial analyte, toa finite reaction zone of said solid inert porous medium so as toeffectively immobilize said analyte within the reaction zone of saidporous medium; (c) applying a labeled compound, consisting essentiallyof an indicator conjugated to an antigen which is the same antigen asthe microbial analyte, to substantially the center of said reaction zoneof said solid inert porous medium under conditions which favor theimmunochemical interaction of said antigen conjugated with an indicatorwith said antibody so as to effectively immobilize said antigenconjugated with an indicator within the reaction zone of the porousmedium; (d) applying a wash solution, comprising an eluting solvent, tosubstantially the center of the reaction zone in a quantity sufficientto effect radial chromatographic separation, within said porous medium,of the unbound labeled compound from that which is bound to saidantibody within the delimited area of the reaction zone; and (e)observing the extent to which the bound labeled compound is presentwithin the delimited area of said reaction zone by measurement of thelevel of indicator in said delimited area.
 18. The assay of claim 17,wherein said antigen conjugated with an indicator is addedsimultaneously with the fluid sample.
 19. The assay of claim 17, whereinsaid antigen conjugated with an indicator is added after the fluidsample.